Is it star activity? - (Oct/23/2007 )
Today I digested one vector with Sda1 restriction enzyme. I know that this vector got only one site for this restriction enzyme. I used 5 unit of enzyme for 1 micro gram of vector and digested for 2.30 hrs. But surprisingly I got 2 fragments, when I got single band with another vector in the same condition. Is it star activity??? Please give me some hints..Thank you
-sijo-
QUOTE (sijo @ Oct 23 2007, 08:00 AM)
Today I digested one vector with Sda1 restriction enzyme. I know that this vector got only one site for this restriction enzyme. I used 5 unit of enzyme for 1 micro gram of vector and digested for 2.30 hrs. But surprisingly I got 2 fragments, when I got single band with another vector in the same condition. Is it star activity??? Please give me some hints..Thank you
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
-aztecan princess-
Thank you very much for your reply. Sorry it was my mistake. Later I found one more site for SdaI. Now I am proceeding with some partial digestion and all. Thank you..
QUOTE (aztecan princess @ Oct 23 2007, 07:50 AM)
QUOTE (sijo @ Oct 23 2007, 08:00 AM)
Today I digested one vector with Sda1 restriction enzyme. I know that this vector got only one site for this restriction enzyme. I used 5 unit of enzyme for 1 micro gram of vector and digested for 2.30 hrs. But surprisingly I got 2 fragments, when I got single band with another vector in the same condition. Is it star activity??? Please give me some hints..Thank you
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
-sijo-
QUOTE (sijo @ Oct 24 2007, 03:04 AM)
Thank you very much for your reply. Sorry it was my mistake. Later I found one more site for SdaI. Now I am proceeding with some partial digestion and all. Thank you..
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
QUOTE (aztecan princess @ Oct 23 2007, 07:50 AM)
QUOTE (sijo @ Oct 23 2007, 08:00 AM)
Today I digested one vector with Sda1 restriction enzyme. I know that this vector got only one site for this restriction enzyme. I used 5 unit of enzyme for 1 micro gram of vector and digested for 2.30 hrs. But surprisingly I got 2 fragments, when I got single band with another vector in the same condition. Is it star activity??? Please give me some hints..Thank you
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
Good luck with the parcial digestion!!! It's not easy, as least in my experiences because the amount of the fragment wanted is small.
-aztecan princess-
Ya I know. But I did successfully 2 times partial digestion earlier and I think I got the trick. So I am now quite confident about partial digestion though it is not so easy. Thank you very much for your suggestion...
QUOTE (aztecan princess @ Oct 24 2007, 11:46 AM)
QUOTE (sijo @ Oct 24 2007, 03:04 AM)
Thank you very much for your reply. Sorry it was my mistake. Later I found one more site for SdaI. Now I am proceeding with some partial digestion and all. Thank you..
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
QUOTE (aztecan princess @ Oct 23 2007, 07:50 AM)
QUOTE (sijo @ Oct 23 2007, 08:00 AM)
Today I digested one vector with Sda1 restriction enzyme. I know that this vector got only one site for this restriction enzyme. I used 5 unit of enzyme for 1 micro gram of vector and digested for 2.30 hrs. But surprisingly I got 2 fragments, when I got single band with another vector in the same condition. Is it star activity??? Please give me some hints..Thank you
Greater than 5-fold overdigestion with SdaI may result in star activity. Or if the concentration of glycerol is more than 10 % in the reaction.
But if you digested just some hours, is doubt it’s start activity, are you sure there is just one cut site?
I’ll re-digest with less enzyme to be sure.
Good luck with the parcial digestion!!! It's not easy, as least in my experiences because the amount of the fragment wanted is small.
-sijo-